This invention relates to a method for the production of bacterial endospores from Pasteuria, or Pasteuria-like, bacteria. These bacteria are able to produce endospores that have the unique and useful property of being able to attach to, infect, grow in, re-sporulate in, and kill certain types of phytopathogenic nematodes and other soil-dwelling nematodes.
The worldwide destructive capacity of nematodes on cultured plants and the consequent loss of crop productivity are well known (Sasser, J. N. [1980] Plant Disease 64(1):36-41). Chemical nematicides, which have been the control method of choice, are rapidly becoming anathematized because of their real and potential adverse impacts on the environment; the need for development of alternative, bio-rational control agents of nematodes has been widely stated.
Endospore-forming bacteria of the Pasteuria group have been publicly recognized as potential biorational control agents of plant-pathogenic nematodes (Sayre, R. M. [1980] Plant Disease 4:527-532; Stirling, G. R. [1984] Phytopathology 74:55-60). The first report of a Pasteuria-like organism infecting nematodes was by N. A. Cobb (2nd ed. Hawaiian Sugar Planters Assoc., Expt. Sta. Div. Path. Physiol Bull., vol. 5, pp. 163-195, 1906), who observed numerous refractile spores in nematodes of the species Dorylaimus bulbiferous. R. M. Sayre et al. (Sayre, R. M., M. P. Starr, A. M. Golde, W. P. Wergin, and B. Y. Endo [1988] Proc. Helminthol. Soc. Wash. 55(1):28-49) suggested that the endospore-forming bacterium Pasteuria penetrans is a specific parasite of the root-knot nematode Meloidogyne incognita and proposed that other bacteria of the "Pasteuria group" deserve separate species designations. Starr et al. (Starr, M. P., and R. M. Sayre [1988] Ann. Inst. Pasteur/Microbiol. 139:11-31) assigned the species name Pasteuria thornei sp. nov. to the endospore-forming bacterium that is primarily parasitic on the root-lesion nematode Pratylenchus brachyurus, and the species name Pasteuria penetrans sensu stricto emend. to the endospore-forming bacterium which is primarily parasitic on the rootknot nematode Meloidogyne incognita.
Although bacteria of the Pasteuria group have a recognized potential for use as biorational control agents against phytopathogenic nematodes, their widespread use in commercial agriculture will depend on the availability of reliable methods for the large-scale production of spores having specificity against the phytopathogenic nematodes of concern to farmers. One method of production would be to induce spore formation following true in vitro, axenic growth of the vegetative phase of any select Pasteuria species on an artificial growth medium consisting of inexpensive, readily available materials. Such systems are not known at this time.
Most of the experimental work with the Pasteuria group of bacteria has used spores produced in live nematodes, cultivated on whole plants in greenhouses where aseptic conditions do not prevail. In two exceptions, Verdeho et al. (Verdeho, S., and R. Mankau [1986] Journal of Nematology 18:635) have reported on the oligoxenic culture of Pasteuria penetrans in live Meloidogyne incognita on excised tomato root culture; and Reise et al. (Reise, R. W., K. J. Hackett, R. M. Sayre, and R. N. Huettel [1988] Abstracts of the 27th Annual Meeting Society of Nematologists, p. 75) have studied factors in various tissue culture media affecting Pasteuria isolates from Heterodera glycines, Meloidogyne incognita, and Pratylenchus brachyurus. Their attempts are directed at a genuine in vitro cultivation of Pasteuria, which attempts fail on the basis of the fundamental criterion that a genuine in vitro cultivation of any prokaryotic organism must be marked by a continual survival and proliferation of the organisms, upon transfer to a fresh medium, at some definable growth rate that is characteristic of the genotype of the organism and the environmental conditions.
Our invention does not rely on the genuine in vitro cultivation of the Pasteuria, but is directed at the production of Pasteuria spores on explanted or cultured nematode tissue. The key difference is that our chosen medium is nematode tissue, from a natural host or alternative host nematode, whose capacity to support Pasteuria growth and subsequent sporulation we have experimentally demonstrated.
More particularly, this invention relates to the production of endospores from the post-vegetative phases of Pasteuria penetrans, Pasteuria thornei, and related Pasteuria-like organisms which have been grown vegetatively on any of a variety of growth substrates consisting of explanted nematode tissues or nematode cell cultures, but in the absence of whole living phytopathogenic nematodes and their usual plant host materials.